ABSTRACT The alarming increase in rates of type 2 diabetes has remained unabated despite efforts to implement changes to diet and other factors that undoubtedly play a role. One obstacle has been inconsistency in dietary recommendations and, in particular, the role of fructose. Although population studies have linked excess fructose consumption to type 2 diabetes and other metabolic disorders, questions remain regarding the role played by fructose, in part because the precise mechanisms underlying these associations have not been elucidated in humans. For example, multiple tracer studies have demonstrated that fructose consumption is associated with increased rates of fatty acid synthesis (de novo lipogenesis [DNL]), but this finding is questioned in view of studies in which very little labeled fructose was actually incorporated into newly synthesized fat. We propose to resolve this paradox by determining whether fructose increases DNL by its conversion to fat (direct effect) or if fructose is instead preferentially directed into synthesis of new sugar (gluconeogenesis [GNG]), while other typical GNG substrates such as pyruvate are redirected into DNL in the presence of fructose (indirect effect). Another paradox centers on how fructose leads to high levels of glucose in the blood following a meal. In this grant we will test the hypothesis that meals high in fructose overwhelm the GNG pathway and lead to increased glucose output into the blood. We further hypothesize that meals with lower fructose content can be tolerated by persons with normal fasting insulin and glucose levels but not by those with pre-diabetes. To address these questions, we will perform four one-day dietary studies in men and women with either pre-diabetes or normal glucose and insulin levels. The study is based on the premise that uncontrolled hepatic fluxes from excess fructose consumption play a key role in lipid- and carbohydrate- associated dysregulation. We will employ a feeding paradigm using defined liquid meals and a combination of oral and intravenous stable (not radioactive) isotopes, in tracer and non-tracer amounts, to simultaneously measure fluxes in the GNG and DNL pathways in response to low or high fructose intake. We will also test whether fluxes in these pathways are altered in pre-diabetic individuals compared to controls with normal glucose and insulin levels. By simultaneously measuring the GNG and DNL pathways, this proposal has the potential to both generate important advances in understanding the dynamics of postprandial fructose metabolism and provide important mechanistic information to support or strengthen evidence-based nutritional recommendations regarding limitations on fructose intake.